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Human health and nutrition - INRA Montpellier

K-1084

Rhiz sphere 2


Keynote presentations

Human pathogens in the rhizosphere

Warriner Keith

University of Guelph, Food Science, University of Guelph N1G 2W1 Guelph, Ontario Canada

Session 17 - Human health and nutrition

The number of foodborne illness cases linked to fresh produce has dramatically increased over the last two decades. In 2006, there were

over 13 outbreaks or product recalls within North America linked to fresh produce. The most notable was the outbreak linked to spinach

contaminated with Escherichia coli O157:H7 that caused 199 clinical cases and 3 deaths. To address the number of foodborne illness outbreaks

there is a need for a deeper understanding on the nature of human pathogen:plant interactions. Hitherto it has been considered that

human pathogens such as Salmonella and E. coli O157:H7 survive relatively poorly outside the host environment. However, accumulating

evidence suggests that human pathogens can persist and become established in the rhizosphere of plants. The objective of the following

presentation is to outline the current knowledge on the dissemination of pathogens in the environment and the nature of human

pathogen:plant interactions. Factors that contribute the persistence of human pathogens in water and soil environments will be outlined.

Current knowledge on the interaction of human pathogens with a range of different plant types will be covered. The potential of human pathogens

to become integrated into the endophytic microflora of plants and implications for food safety will be discusses. Evidence will be presented

on how certain strains of human pathogens have adapted mechanisms to avoid plant defence systems that would ordinarily prevent

invasion by opportunistic saprophytes. The interaction of human pathogens with rhizosphere microflora is a relatively unexplored area. From

studies performed to date it has been identified that certain members of the rhizosphere population exhibit positive effects with others acting

antagonistically. On going research is developing novel biocontrol methods based on competitive microflora applied in conjunction with lytic

bacteriophage to reduce the prevalence of human pathogens within the rhizosphere. Finally an overview of key knowledge gaps will be outline.

Specifically, at present it is unclear why certain pathogens appear adapted to specific plant types and underlying genetic/physiological

attributes that enhance persistence. Such knowledge is vital to develop interventions to enhance the microbiological safety of fresh produce.

K-1092

Detrimental effects of glyphosate on micronutrient acquisition of plants in the rhizosphere

Cakmak Ismail 1 , Ozturk Levent 1 , Eker Selim 2 , Neumann Gunther 3 , Römheld Volker 3

1

Sabanci University, Faculty of Engineering and Natural Sciences, 34956 Istanbul, Turkey

2

Cukurova University Faculty of Agriculture, Department of Soil Science 01330 Adana, Turkey

3

Hohenheim University Institute of Plant Nutrition 70593 Stuttgart, Germany

Glyphosate is a commonly applied global herbicide that is highly phloem mobile and rapidly translocated into growing parts of plants. Data

in literature show that up to 80 % of the foliar-absorbed glyphosate is transported into young shoot parts and roots. Glyphosate and its toxic

metabolite AMPA (aminomethylphosphonic acid) accumulates in root tissues and/or released into rhizosphere at very high concentrations.

The half-life of glyphosate in rhizosphere is long enough to cause adverse effects on microorganisms and plants. There is increasing number

of field observations showing widespread occurrence of micronutrient deficiencies in cropping systems with frequent glyphosate applications.

Recent short-term experiments demonstrated that glyphosate existing in root tissues or rhizosphere results in severe impairments in uptake

and transport of micronutrients, especially iron (Fe) and manganese (Mn). In studies with non-target plants (e.g., sunflower and soybean)

foliar spray of glyphosate significantly reduced the number of Mn-reducing microorganisms in rhizosphere and inhibited Mn acquisition of

plants. Besides Mn, glyphosate also caused significant decreases in Fe and Zn acquisition. Decreases in root uptake of micronutrients were

evident within 48 h after foliar spray of glyphosate. In Fe deficient plants, foliar applied glyphosate for 24 h was effective to inhibit ferric reductase

activity of root cells, while proton release activity was not affected. Glyphosate also showed a marked inhibitory effect on the root-toshoot

transport of micronutrients. It seems likely that glyphosate is antagonistic to uptake and transport of Mn and Fe, most probably by formation

of poorly soluble or stable glyphosate-metal complexes in rhizosphere and plant tissues. Inhibitory effect of glyphosate on Mn reducing

microorganisms is possibly related to impairments in their shikimate pathway. In cropping systems with intensive glyphosate applications,

contamination of non-target plants with glyphosate is a common phenomena caused by glyphosate spray drift or root uptake of glyphosate

residues in soil. Under such conditions a special attention should be paid to micronutrient status of plants, particularly on soils with

low availability of micronutrients.

Session 17

247

International Conference Montpellier - France 26-31 August 2007


P-660

Rhiz sphere 2

Session 17 - Human health and nutrition


Poster presentations

Endophytic colonization of Trifolium repens by Clostridium botulinum

Iwobi Azuka 1 , Schmid Michael 1 , Gessler Frank 2 , Hartmann Anton 1 , Boehnel Helge 2

1

GSF Research Institute for Health and Environment Microbe-Plant Interactions Ingolstaedter Landstrasse 1 85764 Neuherberg Munich Munich

Germany

2

Institute for Applied Biotechnology in the Tropics, University of Goettingen, Goettingen, Germany

The genus Clostridium encompasses a broad spectrum of Gram-positive, obligate anaerobic bacteria. Although clostridia are ubiquitously

distributed in the soil, their lifestyle in plants has not been fully elucidated. It was suggested recently that an animal / human Clostridium botulinum

pathogen, could interact efficiently with clover plants (T. repens), leading even to a plant growth stimulating effect (studies were carried

out in Goettingen). However, a detailed colonization study of Clostridium botulinum was not undertaken until now.

Clover seeds were planted in soil harbouring spores of the non-pathogenic C. botulinum 1739 strain and Rhizobium leguminosarum, the

nodule forming, nitrogen-fixing symbiont of clover. After 4 to 8 weeks, plants were harvested and root sections subjected to FISH (fluorescent

in situ hybridization) analysis, using a Clostridium specific phylogenetic oligonucleotide probe (Chis150-Cy3), in combination with a

general bacterial probe (EUB338mix-Fluos).

FISH-analysis combined with confocal laser scanning microscopic (CLSM) studies demonstrated clostridial cells within the central vascular

cylinder. This indicates that the clostridia can efficiently enter roots, and may be translocated to the aerial parts of the plant. Endophytic clostridia

were also detected in the roots of the clover plants from the experimental field plots in Goettingen, Germany, with preferred localization

observed in the phloem cells. Thus, toxin-producing clostridia may be translocated to the shoots, and thus could potentially intoxicate

grazing livestock.

P-665

Caenorhabditis elegans – screening for potential human pathogens derived from the rhizosphere

Pirker Heidemarie, Zachow Christin, Berg Gabriele

Graz University of Technology, Environmental Biotechnology, Petersgasse 12, A-8010 Graz, Austria

Three different bacteria strains (Pseudomonas fluorescens L13-6-12, Serratia plymuthica 3Re4-18, Pseudomonas trivialis RE*1-1-14), derived

from the rhizosphere and the endorhiza of sugar beet plants, respectively, are identified as antagonists against Rhizoctonia solani causal

agent of the late sugar beet rot. These microorganisms are intended to use for biological control of the plant pathogen in field. To identify

possible human pathogenicity of the three strains, a screening assay with the nematode Caenorhabditis elegans was conducted.

The killing assay was arranged with C. elegans which was grown on minimal medium. The killing occurs over a period of several days and

is referred as slow killing. The slow killing involves an infection-like process and correlates with the accumulation of bacteria within the worm

intestines. The biological control strains were incubated with C. elegans and every 24 h the living and dead worms were scored. Also reproduction

and movements were important factors of observation. All experiments were carried out with Escherichia coli OP 50 as a non pathogenic

feeding source and Pseudomonas aeruginosa as positive control. The human opportunistic pathogen Pseudomonas aeruginosa kills

Caenorhabditis elegans and E. coli is nutrition for the nematode. A tested strain was considered as pathogenic when the percentage of live

worms after two days is ≤ 50%. In conclusion, all tested strains show a death rate of C. elegans ≤ 20% after two days. The results suggest

that the rhizobacteria are harmless to human health and an application as a biological control agent is possible.

248

P-467

Iron and zinc bioavailability from forty-five sorghum genotypes from West Africa

Kayodé Polycarpe 1 , Linnemann Anita 2 , Hounhouigan Joseph 3 , Nout Rob 2 , Van Boekel Tiny 2

1

Université d'Abomey-Calavi Département de Nutrition et Sciences Alimentaires FSA/DNSA/UAC 01BP 526 Cotonou Litoral Benin

2

Dept of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

3

Faculté des Sciences Agronomiques, Université d’Abomey-Calavi, 01 BP 526 Cotonou, Bénin

Iron (Fe) and zinc (Zn) deficiencies constitute a major public health problem in many African countries. They mostly affect infants and pregnant

women and may have serious consequences. Chronic micronutrient deficiencies, particularly of Fe, Zn and vitamin A, cause child mortality,

impaired mental and physical development, decreased work output, and contribute to morbidity from infections (1-3). In semi-arid tropics

worldwide, sorghum is a major staple and one of the main dietary sources of macro- and micronutrients (8). Identification and promotion

of local germplasm with high mineral availability can contribute to the improvement of the micro-nutrient status of the consumers, since

they may have a relatively high chance of adoption by the users compared to newly bred varieties from elsewhere. We determined Fe, Zn

and phytate content of 45 sorghum genotypes grown under the Guinea Savannah climate of West Africa, and evaluated their [phytate]/[Fe]

and [phytate]/[Zn] molar ratios as indices for mineral availability. The aim was to identify varieties with high Fe and Zn availability to consumers.

The Fe content of the grains ranged from 31 to 99 mg/kg with an average of 58 mg/kg. The Zn content ranged from 14 to 38 mg/kg

with an average of 25 mg/kg. The phytate content of the grain ranged from 0.5 to 3.5 % with a mean of 1.2 %. No varieties provide adequate

Zn to meet nutritional requirements of sorghum consumers. Within the local germplasm we detected seven varieties promising adequate Fe

supply in food uses as they showed a [phytate]/[Fe] ratio lower than 14, which is the critical value above which Fe availability is strongly

impaired. These varieties could therefore be recommended for the preparation of food products like porridges, in which processing methods

only have a slight diminishing effect on phytate levels. This may also serve as a basis for the selection and breeding of varieties that can be

used to prepare foods with a high Fe availability.

International Conference Montpellier - France 26-31 August 2007


Rhiz sphere 2

Session 17 - Human health and nutrition

P-1031

Ecological interaction of Escherichia coli O157:H7 with a biocontrol Pseudomonas fluorescens strain producing

2,4-diacetylphloroglucinol.

Fremaux Bastien 1 , Prigent-Combaret Claire 2 , Delignette-Muller Marie-Laure 1 , Pujic P 2 , Moënne-Loccoz Yvan 2 , Vernozy-Rozand Christine 1

1

Unité de Microbiologie Alimentaire et Prévisionnelle, Ecole Nationale Vétérinaire de Lyon, 69280 Marcy l’étoile, France

2

UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, 69622 Villeurbanne, France

Escherichia coli O157:H7 is an emerging foodborne pathogen that can persist extensively in soil, which might be a significant factor of contamination

of cattle and humans. Here, our objective was to assess whether the 2,4-diacetylphloroglucinol (Phl)-producing soil bacterium P.

fluorescens F113 could act as a natural biocontrol agent of E. coli O157:H7, since Phl can inhibit certain bacteria. First, growth inhibition tests

of 73 E. coli O157H7 isolates of diverse origins by P. fluorescens F113 and its Phl-negative biosynthetic mutant F113G22 were performed

on solid complex medium. Inhibition of E. coli O157:H7 by P. fluorescens F113 took place but was of small magnitude, whereas F113G22

did not inhibit the growth of any of the O157:H7 isolates. Accordingly, all E. coli O157:H7 isolates were inhibited by synthetic Phl on plate,

but the minimum inhibitory concentration was rather high (> 128 µg ml -1 ). The 73 E. coli O157:H7 were then characterized by Pulse Field Gel

Electrophoresis (PFGE) and their virulence profiles by PCR on shigatoxin-encoding stx genes. The E. coli O157:H7 isolates that were resistant

to high Phl concentrations were found in three main groups, which correspond to stx1+ stx2+ virulence profile, faeces isolates, and isolates

belonging to one particular PFGE cluster. In addition, both P. fluorescens F113 and Phl-negative derivative are able to limit the growth of E.

coli O157 in sterile soil and to a lesser extent in the wheat rhizosphere. Microscopic observations showed that P. fluorescens F113 was exclusively

recovered on the root surface, but E. coli O157:H7 was also visualized within the plant tissues. In conclusion, Phl-producing P. fluorescens

has the potential to inhibit the survival of E. coli O157:H7 in soil and rhizosphere but Phl is only a minor component of this inhibition

effect.

P-1024

P-1290

Does the rhizosphere of dairy alpine grasslands constitute an environmental reservoir for Escherichia coli ?

Texier Stephanie, Trevisan Dominique, Gourdon Marie-Hélène, Jocteur-Monrozier Lucile, Poulenard Jérôme, Poirier Marie-Andrée, Quétin Philippe,

Moënne-Loccoz Yvan, Faivre Pierre, Dorioz Jean-Marcel, Prigent-Combaret Claire

UMR 5557 CNRS Ecologie microbienne Université Claude Bernard 43 bd du 11 novembre, F-69622, Villeurbanne, France

In pastures, dairy cows release significant amounts of faeces (cow pads). The latter contain high numbers of faecal bacteria including sometimes

potential human pathogens. This may be of particular significance in alpine grasslands, where water runoff is likely to enhance environmental

dissemination of these faecal bacteria. In the case of Escherichia coli, time analysis of stream-water monitoring data led to the

hypothesis that the rhizosphere could serve as reservoir in dairy alpine grasslands, releasing E. coli towards surface water during strong rain

events. Here, this hypothesis was assessed by investigating MPN counts of E. coli (indicator of faecal contamination) in comparison with

those of fluorescent Pseudomonas spp. (indigenous populations). Whereas cow pads contained up to 10 6 -10 7 E. coli per g, the rhizosphere

harboured about 10 4 E. coli per g soil year-round, even after disappearance of cow pads (i.e. end autumn till spring). Differences were found

when comparing distinct pastoral areas (i.e. of contrasted soil physicochemical characteristics and plant community composition) within the

watershed or different rhizosphere soil depths. In comparison, Pseudomonas populations fluctuated more. PCR-DGGE analysis of the E. coli

β-D-glucuronidase gene uidA gave one or two majority bands in cow pads, whereas in the rhizosphere these two bands were associated

with several others of lower intensity. These results indicate that the rhizosphere of dairy alpine grasslands constitutes an environmental

reservoir of E. coli populations, refuting the principle of E. coli as transient environmental survivor and its use as faecal contamination indicator.

This will need to be taken into account when building models of surface water contamination. This study raises also the possibility that

rhizosphere-adapted faecal E. coli could have formed naturalised E. coli subpopulations, which are now part of the indigenous rhizosphere

community of various alpine grassland plant species.

Controled fermentation of green table olives from Picholin moroccain variety.

Lamzira Zahra 1 , Ghabbour Nabil 2 , Thonart Philipe 3 , Markaoui Moustapha 4 , Asehraou Abdeslam 2

1

Université Mohamed Premier, Faculté des Sciences Département de Biologie Laboratoire de Biologie des Plantes et des Microorganismes

60000 Oujda Oujda Morocco

2

Laboratoire LBPM, équipe de Microbiologie Appliquée, Département de Biologie, Faculté des Sciences, Université Mohamed 1er, Oujda,

Maroc.

3

Centre Wallon de Biologie Industrielle, Faculté Universitaires des Sciences Agronomiques, Gembloux, Belgique.

4

Laboratoire de Biochimie, Département de Biologie, Faculté des Sciences, Oujda, Maroc

Effect of different initial brine treatments were studied on fermentation of Picholin moroccain variety inoculated with Lyophilised or fresh culture

of Lactobacillus plantarum, as starter culture on an industrial scale. This strain was isolated from olive brine. pH, acidity, FMAT, Yeast

and mould, Staphylococcus, LAB, Enterobacteria were evaluated during the fermentation. Quality of the finish product was monitored by

quantification of different olive defects. Sensory evaluation of taste, aroma, and color were carried out with 14 members. The found results

showed an improvement of the organoleptic and microbiological quality.

249

International Conference Montpellier - France 26-31 August 2007


Rhiz sphere 2

Session 17 - Human health and nutrition

250

International Conference Montpellier - France 26-31 August 2007

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